Patent Application
20170146519
The innate immune system includes an array of sentinel proteins termed pattern recognition receptors (PRRs) that sense and react to microbe- and danger-associated molecular patterns (reviewed in Broz P et al, Nat Rev Immunol 13, 551-565 (2013); incorporated by reference herein). These patterns are often constituents or replication intermediates of intracellular (especially viral) pathogens. PRRs respond to this engagement by initiating signaling pathways that bring about the expression or processing of cytokines, chemokines, and effector molecules that both directly block microbial replication and facilitate related adaptive immune processes. As such, PRRs represent an essential first line of immunological defense against infection and are the target of both microbial inhibitory phenotypes as well as pharmacologic manipulation for therapeutic purposes (reviewed in Es-Saad S et al, Curr Opin Virol 2, 622-628 (2012); incorporated by reference herein).
Synthesis and secretion of interferon (IFN) proteins is often a primary outcome of PRR mediated signaling. This includes multiple subtypes of IFNα and β (type I IFN) as well as IFN λ1-3 (type III IFN). IFNs act via cognate cell surface receptors by triggering a phosphorylation cascade involving Janus and tyrosine kinases (Jak1, Tyk2) and signal transducer and activator of transcription 1 and 2 (STAT1/2) transcription factors that amplify the expression of antiviral effector and other immune stimulatory genes conventionally termed IFN-stimulated genes (ISGs). PRR-mediated expression of IFNβ is particularly well characterized and requires phosphorylation of the transcription factor IFN regulatory factor 3 (IRF3) by serine kinases TANK Binding kinase 1 (TBK1) and I Kappa B kinase ε (IKKε) (Sharma S et al, Science 300, 1148-1151 (2003); incorporated by reference herein). This occurs primarily through pathways that utilize specific adaptor proteins acting as integration points for upstream PRRs. TIR-domain-containing adaptor-inducing IFNβ (TRIF; also called TICAM1) is required for signals initiated by Toll-like receptors (TLRs) 3 and 4 (Yamamoto M et al, Science 301, 640-643 (2003) and Oshiumi H et al, Nat Immunol 4, 161-167 (2003); both of which are incorporated by reference herein). IFN promoter stimulator 1 (IPS-1; also called MAVS, VISA, or Cardif) is employed by RIG-I and MDA5, that both sense cytoplasmic dsRNA (Xu L G et al, Mol Cell 19, 727-740 (2005); Kawai T et al, Nat Immunol 6, 981-988 (2005); Seth R B et al, Cell 122, 669-682 (2005); Meylan E et al, Nature 437, 1167-1172 (2005); all of which are incorporated by reference herein. Stimulator of IFN genes (STING; also called MITA, TMEM173, MPYS, ERIS) (Ichikawa H et al, Nature 455, 674-678 (2008); Zhong B et al, 29, 538-550 (2008); and Sun W et al, Proc Natl Acad Sci USA 106, 8653-8658 (2009); all of which are incorporated by reference herein) is actually both a PRR for cyclic dinucleotides (CDN) via a binding pocket in its C-terminal cytoplasmic domain (CTD) Burdette D L et al, Nature 478, 515-518 (2012); Sun L et al, Science 339, 786-791 (2013); and Wu J et al, Science 339, 826-830 (2013); all of which are incorporated by reference herein) as well as an adaptor molecule for multiple cytoplasmic receptors of dsDNA (Unterholzner L et al, Nat Immunol 11, 1004 (2010); Stavrou S et al, Cell Host and Microbe 17, 478-488 (2015); and DeFilippis V R et al, J Virol 84, 585-598 (2010); all of which are incorporated by reference herein). Given the importance of these pathways for innate immune activation and antimicrobial protection they have been the focus of broad and intense research aimed at both understanding their physiological effects and harnessing their potential for contributions to immune-based therapeutics.
Given the ability of the IFN system to render cells and tissues refractory to replication of a wide array of virus types as well as its role in coordinating adaptive immune responses, pharmacologic IFN stimulation has been suggested as a broad spectrum antiviral strategy (Ireton R C et al, Antiviral Res 108, 156-164 (2014); Patel D A et al, PLoS ONE doi:10.1371 (2012); Wong J P et al, Vaccine 27, 3481-3483 (2009); Silin D S et al, Curr Pharm Des 15, 1238-1247 (2009); all of which are incorporated by reference herein). Moreover, factors capable of yielding therapeutic effects via activation of IRF3-mediated responses have been identified and biologically validated. This includes agonists of TLRs shown to block replication of some chronic viruses (Wu J et al, Hepatolory 46, 1769-1778 (2007); Isogawa M et al, J Virol 79, 7269-7272 (2005); and Svensson A et al, J Reprod Immunol 74, 114-123 (2007); all of which are incorporated by reference herein) as well as enhance vaccine immunity (reviewed in (Maisonneuve C et al, Proc Natal Acad Sci USA 111, 12294-12299 (2014); incorporated by reference herein). Similarly, stimulation of the RIG-I/MDA5/IPS-1 by synthetic nucleic acids can be employed for antiviral outcomes against diverse acute viruses (Goulet M L et al, PLoS Pathol 9, e1003298 (2013) and Olagnier D et al, J Virol 88, 4180-4194 (2014); both of which are incorporated by reference herein). Intriguingly, two synthetic small molecules, 10-carboxymethyl-9-acridanone (CMA) (Kramer M J et al, Antimicrobial Agents and Chemotherapy 9, 233-238 (1976); incorporated by reference herein) and the chemically unrelated 5,6-dimethylxanthenone-4-acetic acid (DMXAA) (Perera P Y et al, J Immunol 153, 4684-4693 (1994); incorporated by reference herein) are each capable of activating the STING pathway. Both molecules block multiple, even drug-resistant viruses (Guo F et al, Antimicrob Agents Chemother doi:10.1128/AAC.04321-14 (2014); Cheng G et al, Am J Respir Cell Mol Biol 45, 480-488 (2011); Shirey K A et al, J Leukoc Biol 89, 351-357 (2011); all of which are incorporated by reference herein). Intriguingly, DMXAA exhibits other immunotherapeutic effects including vaccine adjuvanticity (Tang C K et al, PLoS ONE 8, e60038 (2013); Blaauboer S M et al, J Immunol 192, 492-502 (2014); both of which are incorporated by reference herein), anti-angiogenic vascular disruption promoting tumor necrosis (Wallace A et al, Cancer Res 67, 7011-7019 (2007) and Jassar A S, Cancer Res 65, 11752-11761 (2005); incorporated by reference herein), and immune-mediated clearance of solid tumors (Corrales L et al, Cell Rep 11, 1018-1030 (2015); incorporated by reference herein). Unfortunately, CMA and DMXAA were found to only function in mouse, not human cells and tissues (Caviar T et al, EMBO J 32, 1440-1450 (2013); Kim S et al, ACS Chem Biol 8, 1396-1401 (2013); and Kim S et al, ACS Chem Biol 8, 1396-1401 (2013); all of which are incorporated by reference herein and thus were not effective in clinical trials. While analogs of cross-specific stimulatory CDNs have been synthesized (Conlon J et al, J Immunol 190, 5216-5225 (2013); incorporated by reference herein), to our knowledge there exists no published biological characterization of novel synthetic molecular entities that activate human STING dependent innate responses, despite the high and multi-pronged therapeutic potential of exploiting this important immunological protein.
Members of the Alphavirus genus include mosquito-transmitted agents that are re-emerging worldwide and can lead to significant morbidity and mortality (reviewed in (Weaver S C et al, Antiviral Res 94, 242-257 (2012); incorporated by reference herein). Among these is Chikungunya virus (CHIKV), which, despite its evolutionary origin in the Old World, is currently experiencing a severe outbreak in the Caribbean, Central, and South America. Since it first arrived in the Western hemisphere in December 2013 over one million suspected and confirmed cases are estimated to have occurred (Johansson M A, Trends Parasitol 31, 43-45 (2015); incorporated by reference herein). CHIKV disease is characterized by severe joint pain that can persist for months to years. Venezuelan Encephalitis virus (VEEV) is a related virus belonging to the New World clade that has experienced numerous outbreaks in South and Central America as well as southern Texas (Zehmer R G et al, Health Serv Rep 89, 278-282 (1974); incorporated by reference herein). VEEV is a much more deadly agent with fatality rates at approximately 20% but that can reach up to 35% in children (reviewed in (Go Y Y et al, Clin Exp Vaccine Res 3, 58-77 (2014); incorporated by reference herein). Currently no FDA-approved antiviral drugs or vaccines exist for either virus. Interestingly, however, both viruses are extremely sensitive to type I IFN (Couderc T et al, PLoS Pathogens, 10.1371/journal.ppat.0040029 (2008); Lukaszewski R A and Brooks T J, J Virol 74, 5006-5015 (2000); Pinto A J et al, J Interferon Res 10, 293-298 (1990); all of which are incorporated by reference herein). Moreover, being RNA-based viruses their infection triggers IRF3/IFN activation via the IPS-1 pathway (White L K et al, J Virol 85, 606-620 (2011); incorporated by reference herein) and as such may not exhibit evasion phenotypes directed at the cytoplasmic DNA-based STING pathway. In light of this pharmacologic activation of IRF3/IFN via STING may represent an efficacious therapeutic strategy. Disclosed herein is the identification and characterization of a small molecule capable of stimulating IRF3 phosphorylation and IFN production in human cells that prevents replication of Alphaviruses. Reverse genetic studies using CRISPR/Cas9-mediated gene editing are also disclosed that show that this molecule requires STING for its innate gene induction and antiviral activity and thus it represents the first synthetic compound definitively capable of activating this pathway in human cells. Moreover, in vivo stimulation of the STING pathway was also shown to prevent replication of CHIKV demonstrating the potential therapeutic application of pharmacologically targeting activation of this protein.
STING is a pattern recognition receptor of cyclic dinucleotides as well as an innate immune adaptor protein that enables signaling from cytoplasmic receptors to the transcription factor interferon regulatory factor 3. Initiation of these pathways leads to the expression of type I interferons and proteins associated with antiviral and antitumor immunity. Small molecules capable of triggering STING-dependent cellular processes are effective at blocking virus replication, enhancing vaccine efficacy, and facilitating an immune response to cancer cells.
Disclosed herein is the first synthetic small molecule capable of activating STING-mediated signaling in human cells. Also disclosed is that exposure of cells to the compound renders them refractory to replication by interferon-sensitive emerging Alphaviruses. In addition, in vivo stimulation of STING dependent activity also blocks viremia of Chikungunya virus. Ultimately this work may lead to the utilization of STING as a target for multiple immune-mediated therapies.
Disclosed herein is the identification and characterization of a small molecule capable of stimulating IRF3 phosphorylation and IFN production in human cells that prevents replication of Alphaviruses. Through reverse genetic studies using CRISPR/Cas9-mediated gene editing it was shown that this molecule requires STING for its innate gene induction and antiviral activity and thus it represents the first synthetic compound definitively capable of activating this pathway in human cells. Moreover, in vivo stimulation of the STING pathway was also shown to prevent replication of CHIKV demonstrating the potential therapeutic application of pharmacologically targeting activation of this protein.
Disclosed are methods of identifying a test compound that is likely to act as an agonist of one or more proteins in the STING pathway. The methods involve contacting a first transfected human cell with the test compound. The first transfected human cell includes an expression vector. The expression vector includes (i) a polynucleotide encoding a human telomerase reverse transcriptase with expression driven by a constitutively active promoter and (ii) a polynucleotide encoding a bioluminescent or fluorescent protein with expression driven by a promoter that promotes expression in the presence of interferon regulatory factor 3. The first transfected human cell expresses STING. The method further involves contacting a second transfected human cell with the test compound. The second transfected human cell includes the same expression vector as the first transfected human cell, but the second transfected human cell does not express STING. Expression of the bioluminescent or fluorescent protein in the presence of the test compound in the first transfected human cell line that is greater than the expression of the bioluminescent or fluorescent protein in the presence of the test compound in the second transfected human cell line is an indication that the test compound is likely to act as an agonist of one or more proteins in the STING pathway.
In embodiments, the methods can further involve the second promoter also promoting expression in the presence of type I interferon. The second transfected human cell line can lack expression of STING due to the excising of the STING gene using CRISPR/Cas9. The bioluminescent or fluorescent protein can be a luciferase. The methods can further involve assessing whether or not the test compound is not likely to induce NF-κB. The methods can further involve use of a test compound that acts as a positive control including a test compound with the formula:
where X2 is aryl or aryl substituted alkyl, and where R1 and R2 are independently H or halo.
Such positive control compounds include a compound with the formula:
Disclosed are compounds with the formula:
where X2 is aryl or aryl substituted alkyl, and where R1 and R2 are independently H or halo. In embodiments, the disclosed compounds include compounds with the formula:
where X2 is aryl. In still further embodiments, X2 can be phenyl or furanyl, and can include compounds with the formula:
Also disclosed are pharmaceutical compositions comprising the above compounds and a pharmaceutically acceptable carrier.
Also disclosed are methods of inhibiting alphavirus replication in a subject. Such methods involve administering the above pharmaceutical composition to the subject, thereby inhibiting the alphavirus replication. In still further embodiments, the pharmaceutical composition comprises the compound designated G10 herein. In still further embodiments, the alphavirus is chikungunya virus.
SEQ ID NO: 1 is a protein sequence of human STING.
SEQ ID NO: 2 is a protein sequence of human telomerase reverse transcriptase.
SEQ ID NO: 3 is a protein sequence of firefly luciferase protein.
SEQ ID NO: 4 is a protein sequence of human regulatory factor 3 protein.
SEQ ID NO: 5 is a nucleic acid sequence of human ISG154.
SEQ ID NO: 6 is a nucleic acid sequence of human ISG156.
SEQ ID NO: 7 is a nucleic acid sequence of human ISG15.
SEQ ID NO: 8 is a nucleic acid sequence of human VIPERIN.
SEQ ID NO: 9 is a protein sequence of human NF-κB.
SEQ ID NO: 10 is a nucleic acid sequence of human IL-8.
SEQ ID NO: 11 is a nucleic acid sequence of human IL-1β.
SEQ ID NO: 12 is a nucleic acid sequence of human MIP-1α.
SEQ ID NOs: 13-32 are oligonucleotide primers.
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.
As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist essentially of, or consist of.” The transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified.
Agonist: An agonist is an agent, such as a small molecule or protein that binds to a protein and causes, enhances, or augments (to a statistically significant degree) a particular biological effect of the protein. Agonists can be naturally occurring or artificially synthesized compounds. For example, an agonist of a protein in the STING pathway is a compound that augments the natural activity of a protein in the STING pathway (either upstream or downstream).
Alkyl: a branched or unbranched saturated hydrocarbon group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A lower alkyl group is a saturated branched or unbranched hydrocarbon having from 1 to 6 carbon atoms (C1-6 alkyl). The term alkyl also includes cycloalkyls. Alkyl also includes substituted alkyls which are alkyl groups wherein one or more hydrogen atoms are replaced with a substituent such as alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ether, ketone, aldehyde, hydroxyl, carboxyl, cyano, amido, haloalkyl, haloalkoxy, or alkoxy. The term alkyl also includes heteroalkyls. A heteroalkyl contains at least one heteroatom such as nitrogen, oxygen, sulfur, or phosphorus replacing one or more of the carbons. Substituted heteroalkyls are also encompassed by the term alkyl.
Aryl: any carbon-based aromatic group including benzene, naphthalene, and phenyl. The term aryl also includes substituted aryls in which one or more of the hydrogens is substituted with one or more groups including alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ether, ketone, aldehyde, hydroxy, carboxylic acid, cyano, amido, haloalkyl, haloalkoxy, or alkoxy. The term aryl also includes heteroaryls in which one or more of the carbons is replaced by a heteroatom. Examples of heteroatoms include nitrogen, oxygen, sulfur, and phosphorous. Substituted heteroaryls are also encompassed by the term aryl.
Bioluminescent proteins or photoproteins: proteins derived from bioluminescent organisms that emit light by the conversion of chemical bond energy to light energy. Examples of such proteins include luciferase from any source including Aqueora victoria, or Phytonis pyralis.
Contacting: Placement under conditions in which direct physical association occurs, including contacting of a solid with a solid, a liquid with a liquid, a liquid with a solid, or either a liquid or a solid with a cell or tissue, whether in vitro or in vivo. Contacting can occur in vitro with isolated cells or tissue or in vivo by administering to a subject.
Control: A reference standard. A control can be a test compound that is known to be an agonist of a protein in the STING pathway (positive control), such as G10. A control can also be a test compound known not to act as an agonist of a protein in the STING pathway, such as the vehicle in which the test compound is provided, otherwise lacking the test compound (negative control).
Derivative: a compound or portion of a compound that is derived from or is theoretically derivable from a parent compound. Within the current disclosure, a derivative exhibits a substantially similar biological effect in the methods disclosed and claimed herein.
Effective Amount: An amount of an agent that is sufficient to generate a desired response such as reducing or eliminating a sign or symptom of a condition or a disease. An effective amount also encompasses an effective amount of a first agent and an effective amount of a second agent administered in combination with the first agent. In some examples, the effective amount of the two combined agents is less than that of either agent when administered alone.
Fluorescent protein: A protein characterized by a barrel structure that allows the protein to absorb light and emit it at a particular wavelength. Fluorescent proteins include green fluorescent protein (GFP) modified GFPs and GFP derivatives and other fluorescent proteins, such as EGFP, EBFP, YFP, BFP, CFP, ECFP, and circularly permutated fluorescent proteins such as cpVenus.
Heterocycle: A chemical group that includes both heteroaryls and heterocycloalkyls. Heterocycles may be monocyclic or polycyclic rings. Exemplary heterocycles include azepinyl, aziridinyl, azetyl, azetidinyl, diazepinyl, dithiadiazinyl, dioxazepinyl, dioxolanyl, dithiazolyl, furanyl, isooxazolyl, isothiazolyl, imidazolyl, morpholinyl, oxetanyl, oxadiazolyl, oxiranyl, oxazinyl, oxazolyl, piperazinyl, pyrazinyl, pyridazinyl, pyrimidinyl, piperidyl, piperidino, pyridyl, pyranyl, pyrazolyl, pyrrolyl, pyrrolidinyl, thiatriazolyl, tetrazolyl, thiadiazolyl, triazolyl, thiazolyl, thienyl, tetrazinyl, thiadiazinyl, triazinyl, thiazinyl, thiopyranyl, furoisoxazolyl, imidazothiazolyl, thienoisothiazolyl, thienothiazolyl, imidazopyrazolyl, cyclopentapyrazolyl, pyrrolopyrrolyl, thienothienyl, thiadiazolopyrimidinyl, thiazolothiazinyl, thiazolopyrimidinyl, thiazolopyridinyl, oxazolopyrimidinyl, oxazolopyridyl, benzoxazolyl, benzisothiazolyl, benzothiazolyl, imidazopyrazinyl, purinyl, pyrazolopyrimidinyl, imidazopyridinyl, benzimidazolyl, indazolyl, benzoxathiolyl, benzodioxolyl, benzodithiolyl, indolizinyl, indolinyl, isoindolinyl, furopyrimidinyl, furopyridyl, benzofuranyl, isobenzofuranyl, thienopyrimidinyl, thienopyridyl, benzothienyl, cyclopentaoxazinyl, cyclopentafuranyl, benzoxazinyl, benzothiazinyl, quinazolinyl, naphthyridinyl, quinolinyl, isoquinolinyl, benzopyranyl, pyridopyridazinyl and pyridopyrimidinyl groups. The term also includes substituted heterocycles, including substituted forms of all the species above.
Label: A label may be any substance capable of aiding a machine, detector, sensor, device, column, or enhanced or unenhanced human eye in differentiating a labeled composition from an unlabeled composition. Labels may be used for any of a number of purposes and one skilled in the art will understand how to match the proper label with the proper purpose. Examples of uses of labels include purification of biomolecules, identification of biomolecules, detection of the presence of biomolecules, detection of protein folding, and localization of biomolecules within a cell, tissue, or organism. Examples of labels include: radioactive isotopes or chelates thereof; dyes (fluorescent or nonfluorescent), stains, enzymes, nonradioactive metals, magnets, protein tags, any antibody epitope, any specific example of any of these; any combination between any of these, or any label now known or yet to be disclosed. A label may be covalently attached to a biomolecule or bound through hydrogen bonding, Van Der Waals or other forces.
One particular example of a label is a protein tag. A protein tag includes a sequence of one or more amino acids that may be used as a label as discussed above, particularly for use in protein purification. In some examples, the protein tag is covalently bound to the polypeptide. It may be covalently bound to the N-terminal amino acid of a polypeptide, the C-terminal amino acid of a polypeptide or any other amino acid of the polypeptide. Often, the protein tag is encoded by a polynucleotide sequence that is immediately 5′ of a nucleic acid sequence coding for the polypeptide such that the protein tag is in the same reading frame as the nucleic acid sequence encoding the polypeptide. Protein tags may be used for all of the same purposes as labels listed above and are well known in the art. Examples of protein tags include chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly-histidine (His), thioredoxin (TRX), FLAG®, V5, c-Myc, HA-tag, and so forth.
A His-tag facilitates purification and binding to on metal matrices, including nickel matrices, including nickel matrices bound to solid substrates such as agarose plates or beads, glass plates or beads, or polystyrene or other plastic plates or beads. Other protein tags include BCCP, calmodulin, Nus, Thioredoxin, Streptavidin, SBP, and Ty, or any other combination of one or more amino acids that can work as a label described above.
Another particular example of a label is biotin. Biotin is a natural compound that tightly binds proteins such as avidin or streptavidin. A compound labeled with biotin is said to be ‘biotinylated’. Biotinylated compounds can be detected with avidin or streptavidin when that avidin or streptavidin is conjugated another label such as a fluorescent, enzymatic, radioactive or other label.
Operably Linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in such a way that it has an effect upon the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous, or they may operate at a distance.
Polynucleotide: a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The term can be used interchangeably with the term ‘nucleic acid.’ A polynucleotide is made up of four bases; adenine, cytosine, guanine, and thymine/uracil (uracil is used in RNA). A coding sequence from a nucleic acid is indicative of the sequence of the protein encoded by the nucleic acid.
Polypeptide: Any chain of amino acids, regardless of length or posttranslational modification (such as glycosylation, methylation, ubiquitination, phosphorylation, or the like). Herein as well as in the art, the term ‘polypeptide’ is used interchangeably with peptide or protein, and is used to refer to a polymer of amino acid residues. The term ‘residue’ can be used to refer to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. Polypeptide sequences are generally written with the N-terminal amino acid on the left and the C-terminal amino acid to the right of the sequence.
Promoter: A promoter may be any of a number of nucleic acid control sequences that directs transcription of a nucleic acid. Typically, a eukaryotic promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element or any other specific DNA sequence that is recognized by one or more transcription factors. Expression by a promoter may be further modulated by enhancer or repressor elements. Numerous examples of promoters are available and well known to those of skill in the art. A nucleic acid including a promoter operably linked to a nucleic acid sequence that codes for a particular polypeptide can be termed an expression vector.
Recombinant: A recombinant nucleic acid or polypeptide has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more naturally occurring sequences. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. A recombinant polypeptide can refer to a polypeptide that has been made using recombinant nucleic acids, including recombinant nucleic acids transferred to a host organism that is not the natural source of the polypeptide.
Subject: A living multicellular vertebrate organism, a category that includes, for example, mammals and birds. A “mammal” includes both human and non-human mammals, such as mice. In some examples, a subject is a patient, such as a patient diagnosed with cancer. In other examples, a subject is a patient yet to be diagnosed with cancer.
Test Compound: A test compound can be any compound that is suspected of or might be an agonist of one or more proteins in the STING pathway. Examples of test compounds include small molecules, proteins, peptides, or other potential therapeutic compounds. A test compound can also be a compound known to be an agonist of the STING pathway that is used as a positive control. A test compound can also be a compound known not to affect the activity of the STING pathway that is used as a negative control.
Treatment: any therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of metastases, an improvement in the overall health or well-being of the subject, or by other clinical or physiological parameters associated with a particular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. A “therapeutic” treatment is a treatment administered after the development of significant signs or symptoms of the disease.
Disclosed are compounds of the formula:
where R1 and R2 are independently H or halo and where X1 is aryl or aryl substituted alkyl.
Also disclosed are compounds of the formula:
where X2 is aryl. In some embodiments, X2 is benzyl, substituted benzyl, 5 membered heterocycle or substituted 5 membered heterocyle. In still further embodiments, X2 is furanyl. Particular examples of the disclosed compounds include:
which is referred to herein as G10;
which is referred to herein as G10-01;
which is referred to herein as G10-02;
which is referred to as G10-03
which is referred to as G10-04;
which is referred to as G10-05.
The compounds disclosed herein may be included in pharmaceutical compositions (including therapeutic and prophylactic formulations), typically combined together with one or more pharmaceutically acceptable carriers (known equivalently as vehicles) and, optionally, other therapeutic ingredients.
Such pharmaceutical compositions can formulated for administration to subjects by a variety of mucosal administration modes, including by oral, rectal, intranasal, intrapulmonary, intravitrial, or transdermal delivery, or by topical delivery to other surfaces including the eye. Optionally, the compositions can be administered by non-mucosal routes, including by intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, intrathecal, intracerebroventricular, or parenteral routes. In other examples, the compound can be administered ex vivo by direct exposure to cells, tissues or organs originating from a subject.
To formulate the pharmaceutical compositions, the compound can be combined with various pharmaceutically acceptable additives. Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like. In addition, local anesthetics (for example, benzyl alcohol), isotonizing agents (for example, sodium chloride, mannitol, sorbitol), adsorption inhibitors (for example, Tween®-80), solubility enhancing agents (for example, cyclodextrins and derivatives thereof), stabilizers (for example, serum albumin), and reducing agents (for example, glutathione) can be included.
When the composition is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w/v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced at the site of administration. Generally, the tonicity of the solution is adjusted to a value of about 0.3 to about 3.0, such as about 0.5 to about 2.0, or about 0.8 to about 1.7. The compound can be dispersed in any pharmaceutically acceptable carrier, which can include a hydrophilic compound having a capacity to disperse the compound, and any desired additives. The carrier can be selected from a wide range of suitable compounds, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (for example, maleic anhydride) with other monomers (for example, methyl (meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives, such as hydroxymethylcellulose, hydroxypropylcellulose and the like, and natural polymers, such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a carrier, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acidglycolic acid) copolymer and mixtures thereof.
Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters and the like can be employed as carriers. Hydrophilic polymers and other vehicles can be used alone or in combination, and enhanced structural integrity can be imparted to the vehicle by partial crystallization, ionic bonding, cross-linking and the like. The carrier can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and films for direct application to a mucosal surface.
The compound can be combined with the carrier according to a variety of methods, and release of the compound can be by diffusion, disintegration of the vehicle, or associated formation of water channels. In some circumstances, the compound is dispersed in microcapsules (microspheres) or nanoparticles prepared from a suitable polymer, for example, 5-isobutyl 2-cyanoacrylate (see, for example, Michael et al., J. Pharmacy Pharmacol. 43, 1-5, (1991)), and dispersed in a biocompatible dispersing medium, which yields sustained delivery and biological activity over a protracted time.
Pharmaceutical compositions for administering the compound can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of the compound can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
In certain embodiments, the compound can be administered in a time release formulation, for example in a composition which includes a slow release polymer. These compositions can be prepared with vehicles that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearate hydrogels and gelatin. When controlled release formulations are desired, controlled release binders suitable for use in accordance with the disclosure include any biocompatible controlled release material which is inert to the active agent and which is capable of incorporating the compound and/or other biologically active agent. Numerous such materials are known in the art. Useful controlled-release binders are materials that are metabolized slowly under physiological conditions following their delivery (for example, at a mucosal surface, or in the presence of bodily fluids). Appropriate binders include, but are not limited to, biocompatible polymers and copolymers well known in the art for use in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues, and do not trigger significant adverse side effects, such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body.
Exemplary polymeric materials for use in the present disclosure include, but are not limited to, polymeric matrices derived from copolymeric and homopolymeric polyesters having hydrolyzable ester linkages. A number of these are known in the art to be biodegradable and to lead to degradation products having no or low toxicity. Exemplary polymers include polyglycolic acids and polylactic acids, poly(DL-lactic acidco-glycolic acid), poly(D-lactic acid-co-glycolic acid), and poly(L-lactic acid-coglycolic acid). Other useful biodegradable or bioerodable polymers include, but are not limited to, such polymers as poly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid), poly(epsilon.-aprolactone-CO-glycolic acid), poly(betahydroxy butyric acid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethyl methacrylate), polyamides, poly(amino acids) (for example, L-leucine, glutamic acid, L-aspartic acid and the like), poly(ester urea), poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides, and copolymers thereof. Many methods for preparing such formulations are well known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). Other useful formulations include controlled-release microcapsules (U.S. Pat. Nos. 4,652,441 and 4,917,893), lactic acid-glycolic acid copolymers useful in making microcapsules and other formulations (U.S. Pat. Nos. 4,677,191 and 4,728,721) and sustained-release compositions for water-soluble peptides (U.S. Pat. No. 4,675,189).
The pharmaceutical compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use. Sterile solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the compound and/or other biologically active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders, methods of preparation include vacuum drying and freeze-drying which yields a powder of the compound plus any additional desired ingredient from a previously sterile-filtered solution thereof. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
Disclosed are methods of treating a subject with an alphavirus infection using combinations of compositions described herein. The compounds can be administered by any appropriate route including orally or parenterally including buccally, sublingually, sublabially, by inhalation, intra-arterially, intravenously, intraventricularly, intramuscularly, subcutaneously, intraspinally, intraorbitally, intracranially or intrathecally.
The administration of a pharmaceutical composition comprising the disclosed compounds can be for prophylactic or therapeutic purposes. For prophylactic and therapeutic purposes, the treatments can be administered to the subject in a single bolus delivery, via continuous delivery (for example, continuous transdermal, mucosal or intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol). The therapeutically effective dosage of the treatments for viral infection can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms or detectable conditions associated with a disease or condition.
An effective amount or concentration of the disclosed combinations of compounds can be any amount of the two compounds administered by themselves alone or in combination with additional therapeutic agents, is sufficient to achieve a desired effect in a subject. The effective amount of the agent will be dependent on several factors, including, but not limited to, the subject being treated and the manner of administration of the compositions. In one example, a therapeutically effective amount or concentration is one that is sufficient to prevent advancement, delay progression, or to cause regression of a disease or condition, or which is capable of reducing symptoms caused by any disease or condition.
In one example, a desired effect is to reduce or inhibit one or more symptoms associated with a disease or condition characterized by alphavirus infection. The one or more symptoms do not have to be completely eliminated for the composition to be effective. For example, a composition can decrease the sign or symptom by a desired amount, for example by at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as compared to how the sign or symptom would have progressed in the absence of the composition or in comparison to currently available treatments.
The actual effective amount will vary according to factors such as the type of alphavirus infection to be protected against/therapeutically treated and the particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like) time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of treatments for alphavirus infection for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response.
An effective amount is also one in which any toxic or detrimental side effects of the compound and/or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects. A non-limiting range for a therapeutically effective amount of treatments for alphavirus infection within the methods and formulations of the disclosure is about 0.0001 μg/kg body weight to about 10 mg/kg body weight per dose for one or both compounds in the combination, such as about 0.0001 μg/kg body weight to about 0.001 μg/kg body weight per dose for one or both compounds in the combination, about 0.001 μg/kg body weight to about 0.01 μg/kg body weight per dose for one or both compounds in the combination, about 0.01 μg/kg body weight to about 0.1 μg/kg body weight per dose for one or both compounds in the combination about 0.1 μg/kg body weight to about 10 μg/kg body weight per dose for one or both compounds in the combination, about 1 μg/kg body weight to about 100 μg/kg body weight per dose for one or both compounds in the combination, about 100 μg/kg body weight to about 500 μg/kg body weight per dose for one or both compounds in the combination, about 500 μg/kg body weight per dose to about 1000 μg/kg body weight per dose for one or both compounds in the combination, or about 1.0 mg/kg body weight to about 10 mg/kg body weight per dose for one or both compounds in the combination.
Determination of effective amount is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease or condition symptoms in the subject. Suitable models in this regard include, for example, murine, rat, rabbit, porcine, feline, non-human primate, and other accepted animal model subjects known in the arts. Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the treatments for hematological malignancies.
A screening methodology for small molecules capable of stimulating innate immune signaling and effector activity in human cells is disclosed. Human fibroblasts were stably transfected with constitutively expressed human telomerase reverse transcriptase (termed THF) as well as luciferase (LUC) from Phytonis pyralis downstream of a promoter element that is reactive to type I IFN-dependent as well as IRF3-dependent transcription (termed THF-ISRE) [18]. Using these cells in a 384-well high-throughput in vitro screening platform we examined 51,632 chemically diverse compounds in duplicate for their ability to significantly stimulate expression of LUC. Sixteen positive control (1000 U/mL IFNβ) and negative control (1% DMSO) LUC readings were obtained for each plate (μP and μN averages, respectively). Readings for individual compounds (R) on a single plate were designated as significant if R>(μP−μN)*0.5.
G10-mediated induction of IFN/IRF3-dependent LUC was confirmed by exposing the transfected human fibroblast them to a range of concentrations of G10. As shown in
Molecular patterns that culminate in IRF3 activation and synthesis of type I IFN often simultaneously induce pathways leading to activation of the transcription factor NF-κB. It was next examined whether G10 also stimulated this response. Telomerized human fibroblasts (THF) were stably transduced with LUC driven by an NF-κB dependent promoter. As shown in
These results indicated that exposure of cells to G10 stimulates the expression of genes that are dependent on IRF3- and/or IFN-dependent signaling. Numerous such genes have been characterized as antiviral effectors that act either via direct molecular or indirect immunological mechanisms (see Schneider W M et al, Ann Rev Immunol 32, 513-545 (2014); incorporated by reference herein). It was therefore examined whether G10 was capable of generating a cellular state refractory to virus replication in vitro, presumably through the initial activity of IRF3. THF cells were pre-exposed for 6 hours to concentrations of G10 that fell well within a nontoxic range (
Since LUC expression in THF-ISRE reporter cells can be activated directly by IRF3 alone or following IFN-mediated Jak/STAT1/2 signaling it was next examined whether either or both transcription complexes were required for this effect. Derivative THF-ISRE cells were developed from which either the STAT1 or IRF3 protein was stably removed via disruption of the respective coding regions by lentivirus-delivered CRISPR/Cas9 components (Sanjana N E et al, Nat Meth 11, 783-784 (2014); Sternberg S H et al, RNA 18, 661-672 (2012); Mali P et al, Science 339, 823-826 (2013); and Ran F A et at Nat Protoc 8, 2281-2308 (2013); all of which are incorporated by reference herein). As shown in
Since transcription of a reporter gene by G10 does not occur in the absence of IRF3 this strongly implies that G10 stimulates the activation of IRF3, which involves phosphorylation of C-terminal serine residues and subsequently allows its dimerization, nuclear translocation and DNA binding. To verify that IRF3 activation does occur in response to G10 IB was performed using an antibody reactive to phosphorylated IRF3 residue S386 with whole cell lysates harvested from THF exposed to G10 or control stimuli. As shown in
An array of PRRs reacting with multiple classes of pathogen-associated molecules is capable of initiating signaling pathways that terminate in IRF3 activation. As discussed above these conventionally employ an adaptor protein to activate the IRF3-directed kinases TBK1 and IKKε. IFNβ promoter stimulator 1 (IPS-1, also called MAVS) is utilized by RIG-I and MDA5, cytoplasmic sensors of (typically virus-associated) dsRNA. In an effort to characterize the cellular pathway targeted by G10 it was first asked whether IPS-1 is important for the molecule's effect on innate cellular activation. Lentivirus-delivered CRISPR/Cas9 was used to construct THF cells lacking the protein. As shown in
It was then examined whether the IRF3-terminal adaptor protein STING is critical to G10 mediated innate activation. THF-ISRE cells were constructed from which the STING protein is eliminated via CRISPR/Cas9-mediated gene disruption as described above. Knockout of the protein was confirmed visually by IB of whole cell lysates and functionally by demonstrating the absence of IRF3 S386 phosphorylation following treatment with UV-CMV or transfection with 2′3′-cGAMP, both of which are STING dependent cellular reactions (
Based on our observations above that G10-elicited anti-Alphaviral activity requires IRF3 (
STING behaves as a PRR of cyclic dinucleotides (CDN) by way of a direct interaction between them and the protein's C-terminal (ligand-binding) domain (Burdette D L and Vance R E, Nat Immunol 14, 19-26 (2013); incorporated by reference herein). It was therefore asked whether G10 was a direct ligand of human STING, similar to the mouse STING345 specific small molecules DMXAA (Gao P et al, Cell 154, 748-762 (2013) and Prantner D et al, J Biol Chem 287, 39776-39788 (2012); both of which are incorporated by reference herein) and CMA, and sought evidence supporting this hypothesis. Differential scanning fluorimetry was used to examine changes in thermal stability of purified STING-CTD in the presence of G10. Thermal stability of the protein is expected to increase with binding affinity of protein-ligand complexes (Niesen F H et al, Nat Protoc 2, 2212-2221 (2007) and Zhang X et al, 51, 226-235 (2013); both of which are incorporated by reference herein). However, as shown in
STING also behaves as an adaptor molecule [68] required for activating IRF3-targeting kinases by multiple upstream cytoplasmic DNA-sensing PRRs including ZBP1/DAI (DeFilippis V R et al, J Virol 84, 585-598 (2010); incorporated by reference herein), IFI16, DDX41 (Parvatiyar K et al, Nat Immunol 13, 1155-1161 (2012); incorporated by reference herein and IFI203. Given that evidence of direct interaction was not found with STING it is possible that G10 engages one or more of these (or an as yet unknown; see Motani K et al, J Immunol 194, 4914-4923 (2015); incorporated by reference herein) PRRs to initiate STING-dependent activity. In light of this it is interesting to note that G10 does not induce IRF3 activation or IRF3-dependent gene expression in the immortalized promonocytic cell line THP-1 despite the fact that these cells express phenotypically active STING (Li Z et al, PLoS Pathol 11, e1004783-26 (2015); Zhang Z et al, Nat Immunol 12, 959-965 (2011); Jakobsen M R et al, Proc Natl Acad Sci USA 110, E4571-E4580 (2013); Mankan A K et al, EMBO J 33, 2937-2946 (2014); Sun C et al, J Immunol 194, 1819-1831 (2015); and Panchanathan R et al, Innate Immunity 20, 751-759 (2014); all of which are incorporated by reference herein) (
Our data indicate that G10 induces expression of cellular antiviral effector genes and that this process ultimately requires IRF3 and STING. However, transcription of these genes (ISG15, Viperin, ISG54, ISG56) can be triggered in response to either activated IRF3 or IFN-dependent (Jak/STAT) signaling (Grandvaux X et al, J Virol 76, 5532-5539 (2002); Elco C P et al, J Virol 79, 3920-3929 (2005); and Noyce R S et al, J Virol 80, 226-235 (2006); all of which are incorporated by reference herein). It was first examined whether G10 is able to stimulate expression of type I or III interferons, both of which are known to induce Jak/STAT-dependent signaling via type I and type III IFN receptor complexes, respectively. As shown in
Numerous subtypes of type I and III interferons exist 393 and thus demonstrating the presence of secreted molecules requires type-specific immunoassays (ELISA). Secreted interferon of all subtypes was examined using a cell-based reporter assay that reacts with any bioactive type I or III interferon species. For this we utilized THF-ISRE-ΔIRF3 cells described above (
G10 exposure elicits secretion of bioactive IFN in cells that contain STING and IRF3 (
Our observations indicating that STING is required for G10-mediated induction of IRF3-dependent cellular activity led us to examine how the response to the molecule compares to that of a canonical STING ligand such as 2′3′-cGAMP. For this the kinetics of IRF3 phosphorylation and levels of IRF3-dependent gene induction were examined following exposure to each of the molecules. As shown in
To this point our examination of G10-mediated innate activation has focused on human fibroblasts that, while not strictly immortalized, are life-extended through the introduction of telomerase reverse transcriptase. To determine whether G10 is immunostimulatory to a similar degree in more physiologically relevant primary cell types the transcription of IRF3-, IFN-, and NF-κB-dependent genes in human peripheral blood mononuclear cells (PBMCs) was examined. As shown in
While G10-induced innate immune activation is observed in multiple human cell types, similar activity was not detected in murine myeloid-derived RAW264.7 cells. Multiple molecular analogs of G10 were thus constructed in an attempt to identify one that is active in both human and mouse cells. While this allowed characterization of essential and nonessential moieties within the molecule (
DMSO or DMXAA (25 mg/kg) were administered to mice intraperitoneally at 3 h pre-infection with CHIKV (1000 PFU). As shown in
Pharmacologic activation of STING-dependent signaling represents a potentially high impact therapeutic strategy with applications in diverse clinical areas such as broad spectrum antivirals, vaccine adjuvants, vascular disruption, and antitumor immunology. This is represented by multiple successes of the utilization of this approach in mouse models of virus infection (Taylor J L et al, J Infect Dis 142, 394-399 (1980); incorporated by reference herein), enhancement of vaccine immunogenicity (Dubensky T W et al, Ther Adv Vaccines 1, 131-143 (2013); Li X D et al, Science 341, 1390-1394 (2013); and Hanson M C et al, J Clin Invest 125, 2532-2546 (2015); all of which are incorporated by reference herein), immune-mediated tumor necrosis (Peng S et al, J Biomed Sci 18, 21 (2011); incorporated by reference herein), and inhibition of solid tumor angiogenesis (Ching L M et al, Br J Cancer 86, 1937-1942 (2002); Baguley B C and Ching L M, Intl Radiat Oncol Biol Phys 54, 1503-1511 (2002); both of which are incorporated by reference herein). Unfortunately, synthetic small molecules identified thus far have only exhibited suitable efficacy in mouse models due to their strict specificity for the murine STING ortholog. High-throughput screening was used to identify a novel compound (G10) capable of triggering IRF3/IFN-dependent responses and subsequently blocking replication of CHIKV, VEEV, and SINV in human cells. Follow-up work seeking to pinpoint cellular targets essential to the phenotypic responses utilized a reverse genetics approach by way of CRISPR/Cas9-mediated genome editing. This enabled identification of the STING protein as required for G10's biological activity thus indicating that the compound is the first described human-specific synthetic small molecule STING agonist.
G10 triggers innate immune responses that involve expression of IRF3-dependent genes including type I and III interferons. This was observed in telomerized foreskin fibroblasts as well as primary cells such as PBMCs and endothelial cells. Unexpectedly, however, G10 did not induce expression of genes associated with the activity of NF-κB in fibroblasts even though such genes were induced in PBMCs and endothelial cells. Given the central role of NF-κB in generation of pro-inflammatory states that can lead to pathogenic consequences, especially under chronic circumstances, (reviewed in DiDonato J A et al, Immunol Rev 246, 379-400 (2012) and Tonatore L et al, Trends Cell Biol 22, 557-566 (2012); both of which are incorporated by reference herein), it is perhaps desirable that the activity of G10 is more transcriptionally focused to IRF3-dependent responses in certain cell types. It is also interesting that G10 induces type I IFN synthesis in the absence of detectable NF-κB activity given the reported requirement of the transcription factor for this process (Bartlett N W et al, EMBO Mol Med 4, 1244-1260 (2012); Falvo J V et al, Mol Cell Biol 20, 4814-4825 (2000); Wang J et al, J Immunol 185, 1720-1729 (2010); and Panne D et al, Cell 129, 1111-1123 (2007); all of which are incorporated by reference herein). Activation of noncanonical NF-κB subunits may play a role in this case. Undertaking a more thorough molecular investigation of NF-κB subunit activation (e.g. nuclear localization, phosphorylation, DNA binding) will be required to understand this with greater clarity. Importantly, G10 induced the phosphorylation of IRF3 and the protein's deletion led to elimination of reporter gene transcription as well as the compound's anti-Alphaviral activity. As such the innate biological effects of G10 examined here require IRF3-driven gene expression.
Deletion of the adaptor molecule IPS-1/MAVS did not eliminate G10-induced IRF3 phosphorylation or affect the molecule's antiviral effect (
Our results clearly establish an essential role for the signaling molecule STING. Deletion of the STING protein resulted in complete inactivation of G10-mediated IRF3 phosphorylation, IRF3-dependent transcription, IFN secretion, and antiviral activity (
Given that G10 stimulates innate cellular effects that require STING, it was decided to compare the dose dependence of these effects to 2′3′-cGAMP, an established STING ligand. Our results indicate that while G10 may trigger earlier IRF3 phosphorylation than 2′3′-cGAMP, perhaps due to its smaller size and cell permeability, it triggers levels of IRF3-dependent gene expression with overall less potency than 2′3′-cGAMP (
G10 induces synthesis and secretion of bioactive type I and III IFNs and generates an antiviral state in fibroblast cells positive for STING, IRF3, and STAT1 proteins. Based on these results our model for the elicitation of anti-Alphaviral activity by G10 first involves STING-dependent induction of IRF3 followed by IRF3-mediated synthesis and secretion of type I and III IFNs and subsequent IFN-stimulated, STAT1-dependent expression of antiviral effectors. Detection of STAT1-independent ISG expression in response to IFN exposure and IFN-inducing stimuli was unexpected but not unprecedented and has been reported in multiple studies (Hahm B et al, Immunity 22, 247-257 (2005) and Ousman S S et al, J Virol 79, 7514-7527 (2005); both of which are incorporated by reference herein. Blaszcyk and colleagues attribute this to IFN-induced transcriptional complexes composed of IRF9 and STAT2 homodimers although homo- and heterodimers of other Jak/Tyk2-phosphorylated STAT proteins may also play roles (reviewed in Brierly M M and Fish E N, Interferon Cytokine Res 25, 733-744 (2005). Interestingly, IFNβ was able to stimulate some antiviral activity in cells lacking STAT1 and to a degree that varied between viruses with SINV replication being undetectable. The full assortment of STAT1-independent ISGs expressed cannot be inferred from two proteins (Mx2 and ISG56) and as such the differential susceptibilities of CHIKV, VEEV, and SINV to ISG-encoded proteins in general cannot be known based on these results. Yet it is clear that SINV is highly sensitive to STAT1-independent ISGs relative to the other Alphaviruses. Intriguingly, however, while other IRF3-activating, IFN-inducing stimuli were capable of triggering expression of Mx2 and ISG56 in the absence of STAT1, G10 was not. This likely explains the reliance on STAT1 of G10-mediated anti-Alphaviral activity. Why this disparity in STAT1-dependence occurs between SeV, UV-CMV, and G10 is not clear. It is possible that each stimulus triggers the secretion of unique signatures of type I and type III IFN subtypes that subsequently elicit distinct gene expression patterns (Hilkens C M U et al, J Immunol 171, 5255-5263 (2003) and Moll H P et al, Cytokine 53, 52-59 (2011); both of which are incorporated by reference herein). Elucidation of the importance of the various IFN proteins in G10's antiviral effects will require more detailed examination, for instance by comparative transcriptomics, by using subtype-specific neutralizing antibodies or reverse genetics via gene editing.
While the majority of our investigation employed fibroblast cells, it is evident that G10 elicited innate immune activation in primary human cells such as PBMC's (
Unfortunately G10 was unable to stimulate similar activation in murine cells. As such, evaluating the in vivo efficacy of G10 using a well-established mouse model of Alphavirus (CHIKV) infection was not directly practical. Yet IFN-inducing STING agonists (e.g. DMXAA, CMA) have been described that are murine specific. It was therefore examined whether DMXAA triggers IRF3 activation and IRF3-dependent gene induction in a manner comparable to G10. While comparisons of absolute responses are complicated by the fact that different species, cell types, and reagents are employed, DMXAA does trigger rapid IRF3 phosphorylation and dose-dependent IFNβ and ISG transcription in mouse cells (
In summary a synthetic small molecule capable of inducing expression of type I and III IFNs as well as IFN-dependent antiviral effector genes was identified. Using a reverse genetics approach based on CRISPR/Cas9-mediated genome editing to identify cellular targets of the molecule it was shown that this effect requires STING, IRF3, and STAT1 proteins. These molecules are likewise essential to the ability of G10 to elicit a cellular state refractory to replication of Alphavirus species. Furthermore, given the pivotal role of STING it was also shown that pharmacologic activation of the molecule represents an effective anti-Alphaviral strategy in vivo. Given the demonstrated role of STING pathway stimulation in numerous immunological processes, it is being pursued as a therapeutic target for many diseases. Our work demonstrates the feasibility of identifying molecules that activate STING-dependent signaling and yield therapeutic outcomes as well as a strategy for characterizing cellular effects and essential modulatory proteins via genome editing.
Dimenthyl sulfoxide (DMSO) was obtained from Thermo-Fisher. Puromycin was obtained from Clontech and used at 3 μg/mL in cell culture medium. Lipopolysaccharide (LPS) and polybrene were obtained from Sigma-Aldrich. Human recombinant IFNβ and tumor necrosis factor α (TNFα) were obtained from PBL. ONE-Glo cell lysis/luciferin reagent was obtained from Promega. Lucia luciferin reagent was obtained from Invivogen. Lipofectamine LTX was obtained from Life Technologies. Poly(I:C) was obtained from Amersham (27-4729). 2′3′-cGAMP and ppp-dsRNA were purchased from Invivogen (tlrl-cga23 and tlrl-3prna, respectively). Unless otherwise indicated cells were exposed to ppp-dsRNA at 12.5 μg/mL based on a dose response of innate immune activity performed on THF cells. Stocks of G10 were purchased from ChemDiv. DMXAA was purchased from ApexBio. Antibodies used against the following antigens are indicated in parentheses: GAPDH (Santa Cruz SC-51906); STAT1 (Santa Cruz SC-346) IRF3 (Santa Cruz SC-9082); human S386 phospho-IRF3 (Epitomics 2562-1); mouse S396 phospho-IRF3 (cell Signaling 4947); STING (Cell Signaling 3337); IPS-1 (Bethyl A300-782A); IFIT1/ISG56 (Thermo Fisher PA3 848); and Mx2 (Sigma HPA030235).
Human foreskin fibroblasts originally obtained from the American Type Culture Collection were stably transduced with constitutively expressed human telomerase reverse transcriptase and the IRF3/IFN-responsive pGreenFire-ISRE lentivector and were maintained in DMEM containing 10% fetal calf serum (FCS) and antibiotics. Vero, BHK-21, and C6/36 cells were obtained from Alec Hirsch (Oregon Health and Science University) and were grown as described. RAW264.7 cells were obtained from Jay Nelson (Oregon Health and Science University) and transduced with a lentivector that contains firefly luciferase under the control of the type I IFN responsive element obtained from SA Biosciences. THP1-ISG Lucia cells were obtained from Invivogen and maintained in RPMI containing 10% FCS and antibiotics. These cells were differentiated in 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h before stimulation. Human peripheral blood mononuclear cells were obtained from StemCell Technologies and maintained in RPMI containing 10% FCS and antibiotics. Human umbilical microvascular endothelial cells were obtained from Patrizia Caposio (Oregon Health and Science University) and maintained as described in Botto S et al, Blood 117, 352-361 (2011); incorporated by reference herein. All cells were grown at 37° C. and 5% CO2. Sendai virus (SeV) was obtained from Charles River Laboratories and used at 16 HA units/mL. Cytomegalovirus was grown, titered, UV-inactivated, and exposed to cells as described previously (DeFilippis V R et al, J Virol 84, 8913-8925 (2010) and DeFilippis V R et al, J Virol, 80, 1032-1037 (2006); both of which are incorporated by reference herein. West Nile Virus (WNV) was obtained from Alec Hirsch (Oregon Health and Science University) and used as described in Hirsch A J et al, J Virol 79, 11943-11951 (2005); incorporated by reference herein. Vaccinia Virus (VACV) strain Western Reserve was obtained from Klaus Frith (Oregon Health and Science University) and used as described in Alzhanova D et al, Cell Host Microbe 6, 433-445 (2009); incorporated by reference herein. Sindbis virus (SINV) strain Ar-339 was obtained from ATCC. Venezeulan encephalitis virus (VEEV) strain TC83 and Chikungunya virus (CHIKV) strain MH56 were obtained from Michael Diamond (Washington University). CHIKV was derived from an infectious clone as follows. RNA was transcribed from the linearized clone using the T7 mMessage mMachine kit (Ambion) and transfected using Lipofectamine LTX into BHK-21 cells. Resultant virus was propagated in C6/36-insect cells for 48 h to produce high titer viral stocks after pelleting through a 20% sucrose cushion by ultracentrifugation (22,000 rpm, 825206 g for 1.5 hrs). In all cases infectious virus was quantified by serial dilution plaque assays on Vero cells with a carboxymethylcellulose overlay. Unless otherwise indicated experimental infections were carried out in triplicate using a multiplicity of infection (MOI) of 1 plaque forming unit (PFU) per cell. Cell viability was examined by quantitating ATP using the Cell Titer GLO assay according to the manufacturer's instructions (Promega).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) immunoblots were performed as follows. After trypsinization and cell pelleting at 2,000×g for 10 min. whole-cell lysates were harvested in 2% SDS lysis buffer (50 mM Tris-HCl, 20% glycerol). Lysates were electrophoresed in 8% polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore) using semidry transfer at 400 mA for 1 h. The blots were blocked at room temperature for 2 h or overnight using 10% nonfat milk in 1×PBS containing 0.1% Tween 20. The blots were exposed to primary antibody in 5% nonfat milk in 1×PBS containing 0.1% Tween 20 for 18 h at 4° C. The blots were then washed in 1×PBS containing 0.1% Tween 20 for 20, 15, and 5 min, followed by deionized water for 5 min. A 1 h exposure to horseradish peroxidase conjugated secondary antibodies and subsequent washes were performed as described for the primary antibodies. The antibody was visualized using enhanced chemiluminescence (Pierce).
Total RNA was isolated from cells and DNased using a DNA Free RNA Isolation kit according to the manufacturer's protocol (Zymo Research) and quantified by UV spectrometry. Single-stranded cDNA for use as a PCR template was made from total RNA using random hexamers to prime first-strand synthesis by Superscript III reverse transcriptase (Life Technologies) as described in the manufacturer's protocol. Comparison of mRNA expression between samples (e.g., treated versus untreated) was performed using semiquantitative real-time RT-PCR (qPCR) with the Applied Biosystems sequence detection system according to the MCT method (Livak K J and Schmittgen T D 25, 402-408 (2001); incorporated by reference herein). For IFNβ, IFNλ1, and mouse and human GAPDH (housekeeping gene) pre-validated PrimeTime FAM qPCR primer/probe sets obtained from IDT were used. For all other genes Maxima SYBR Green qPCR master mix (Thermo Fisher) was used. Primers for human ISG15, ISG56, ISG54, and Viperin were described in DeFilippis V and Frueh K, J Virol 79, 6419-6431 (2005); incorporated by reference herein).
NF-κB responsive luciferase reporter cells were made using a commercially available replication incompetent lentivirus (Qiagen). Telomerized human fibroblasts were exposed to virus inoculum in the presence of DMEM plus 5 μg/mL polybrene and rocked at 37° C. for 8 h. At two days post inoculation cells were exposed to 3 μg/mL puromycin. After cells were fully resistant to puromycin they were verified for responsiveness to NF-κB-inducing stimuli (e.g. TNFα, SeV, LPS). Genome editing using lentivector-mediated delivery of CRISPR/Cas9 components was performed generally as described previously [56]. Briefly, 20 nt guide RNA (gRNA) sequences targeting protein-coding regions were inserted into the lentiCRISPRv2 vector (AddGene #52961). These sequences are as follows.
Lentivirus was made by transfecting specific lentiCRISPRv2 plasmid along with packaging (psPAX2; AddGene #12260) and VSV-G pseudotyping (pMD2.G; Addgene #12259) plasmids into Lenti-X 293T cells (Clontech) using Lipofectamine-LTX (Life Technologies). Media was harvested at 48 h and 72 h post transfection, centrifuged (3,000×g for 10 min.) and filtered through a 0.45-μm-pore-size filter to remove cell debris. Subconfluent target cells were exposed to lentivirus for 8 h in the presence of 5 μg/mL polybrene. After the cells reached confluence they were split into DMEM plus 10% FCS containing 3 μg/mL puromycin. Transduced cells were passaged in the presence of puromycin for 7-10 days before protein knockout was examined by immunoblot. Cells were next serially diluted twice in 96 well plates to obtain oligoclonal lines purified for gene deletion. Protein knockout was additionally verified functionally by measuring phenotypic responsiveness to relevant stimuli as discussed below.
Confluent reporter cells were plated at 20,000 (THF-ISRE) or 100,000 (THP1-ISG-Lucia) cells per well in a white 96 well plate 24 h before stimulation. Treatments were performed in quadruplicate in 50 μL DMEM plus 2% FCS for 7 h unless otherwise indicated. One-GLO lysis/luciferin reagent (Promega) was added at 1:1 to each well and luminescence measured on a Synergy plate reader (BioTek).
Coding sequences for human STING-C-terminal domain (CTD; AA 137-379) and mouse STING-CTD (AA 137-378) were cloned into pRSET-B vector (Invitrogen) and contained a 6×HIS tag for protein expression in E. Coli strain BL21 (DE3)pLysS (Promega). Sequences were verified before transforming bacteria, which were then grown in LB media 813 at 37° C. until the OD600 reached 0.8. Protein expression was induced with 1 mM IPTG at 16° C. for 18 h. After induction, the culture was centrifuged and the pellet resuspended in 50 mM NaH2PO4, 150 mM NaCl (pH 7.5) and 10% glycerol after which the cells were lysed by sonication. The recombinant soluble STING-CTD was purified by nickel-affinity chromatography (Clontech laboratories) after which it was further purified by gel-filtration chromatography using a HiPrep 16/60 Sephacryl S-100 HR column (GE Healthcare Life Sciences). Protein was eluted in 50 mM NaH2PO4, 150 mM Nacl (pH 7.5) and the eluted fractions containing STING-CTD concentrated using an Amicon centrifugal filter (10 Kd molecular weight cut-off; Millipore). Aliquots of concentrated STING-CTD were immediately stored at −80° C. For thermal shift assay, 1 μg of recombinant human or mouse STING-CTD was used combined with various concentrations of G10, 2′3′-cGAMP, or DMXAA along SYPRO Orange dye (1:1000 dilution) in a 204 reaction (in triplicate). A StepOne Plus Real-time PCR system was used to acquire fluorescence. The samples were subjected to a temperature gradient of 25 to 99° C. The melting curves were plotted and Tm values determined by fitting the curves to Boltzmann sigmoidal equation using the GraphPad Prism 6 software. Three independent experiments were performed.
C57Bl/6J mice (5-7 weeks of age, Jackson Laboratories) were housed in cage units in an animal BSL3 facility, fed ad libitum, and cared for under USDA guidelines for laboratory animals. 25 mg/kg DMXAA (or DMSO alone) was prepared in 50 μL DMSO and injected intraperitoneally. Mice were challenged with 1000 PFU CHIKV via footpad injection in 20 μL RPMI under isoflurane induced anesthesia. Animals were euthanized at 72 h post infection by isoflurane overdose. Blood was collected by cardiac puncture and serum viral loads titered on Vero cells in duplicate as described above.
Methyl 3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxylate (1) was prepared according to a literature procedure (Trifilenkov et al, J. Comb. Chem. 2006, 8, 469-479; incorporated by reference herein).
Methyl 4-(2-chloro-6-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxylate (2). To a solution of compound 1 (670 mg, 3 mmol), potassium carbonate (6 mmol, 830 mg), and 18-Crown-6 (0.3 mmol, 79 mg) in 15 mL of dry acetonitrile was added 6-chloro-2-fluoro-benzylbromide (4.48 mol, 1.0 g). The resulting solution was heated to 70° C. for 24 hours and concentrated in vacuo. The residue was resuspended in chloroform and washed with water and brine, dried over anhydrous sodium sulfate and concentrated in vacuo to give a crude light-brown residue, which was used without further purification.
4-(2-Chloro-6-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxylic acid (3). To a solution of crude compound 2 in 10 mL of THF and 10 mL of EtOH was added 15 mL of 2 M aqueous sodium hydroxide solution (6 mmol). The resulting solution was heated to 60° C. for 1 hour and concentrated in vacuo. The residue was resuspended in water and diethyl ether. The aqueous layer was separated (product) and acidified to pH ˜2 with 1 M aqueous hydrochloric acid solution. Ethyl acetate was added, and the organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. Purification by column chromatography (0% to 10% methanol in dichloromethane) afforded the title compound 3 as a white solid (400 mg, 71% yield over two steps).
4-(2-Chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (G-10). To a solution of compound 3 (1.14 mmol, 400 mg), ethylcarbodiimide hydrochloride (1.71 mmol, 330 mg) and 4-dimethylaminopyridine (1.71 mmol, 210 mg) in 15 mL dry dimethylformamide was added furfuryl amine (1.71 mmol, 0.15 mL). The resulting solution was stirred at room temperature for 10 minutes and to it was added dry triethylamine (3.42 mmol, 0.48 mL). The reaction vessel was flushed with argon, and the reaction was allowed to stir at room temperature overnight. The reaction was diluted with ethyl acetate and washed with 0.5 M hydrochloric acid solution, water, saturated sodium bicarbonate solution, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. Purification by column chromatography (0% to 20% ethyl acetate in dichloromethane) afforded the title compound G-10 as a yellow solid (296 mg, 63% yield).
This application claims the benefit of U.S. Provisional Patent Application 62/258,339, entitled STING AGONISTS, filed on 20 Nov. 2015, and which is incorporated by reference herein.
Work leading to this invention was supported by the United States government under the terms of grant numbers U54 AI081680, U19 AI 109680, and HHSN272201400055C, all of which were awarded by the National Institutes of Health. The United States government has certain rights to this invention.
| Number | Date | Country | |
|---|---|---|---|
| 62258339 | Nov 2015 | US |
